ES2535835T3 - Compositions for the preparation of mature dendritic cells - Google Patents

Abstract

A method for in vitro maturation of at least one immature dendritic cell, which comprises stimulating said immature dendritic cell with TNFα, IL-1β, IFNγ, an agonist of TLR7 / 8 and prostaglandin E2 (PG).

Description

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DESCRIPTION

Compositions for the preparation of mature dendritic cells

The invention relates to new compositions for the preparation of mature dendritic cells, as well as methods for in vitro maturation of immature dendritic cells and the therapeutic uses of dendritic cells that can be obtained by the method of the invention.

Dendritic cells (hereinafter DC by the English expression Dentritic Cells) have a high potential in a patient as adjuvants in the induction of killer cells and tumor-specific helpers (Schuler G, Schuler-Thurner B, Steinman RM. The use of dendritic cells in cancer immunotherapy Curr. Opin. Immunol. 2003 Apr; 15 (2): 138-47. Review. Banchereau J, Palucka AK. Dendritic cells as therapeutic vaccines against cancer. Nat. Rev. Immunol. 2005 Apr; 5 (4): 296-306 Review Salcedo M, Bercovici N, Taylor R, Vereecken P, Massicard S, Duriau D, Vernel-Pauillac F, Boyer A, Baron-Bodo V, Mallard E, Bartholeyns J, Goxe B , Latour N, Leroy S, Prigent D, Martiat P, Sales F, Laporte M, Bruyns C, Romet-Lemonne JL, Provided JP, Lehmann F, Velu T. Vaccination of melanoma patients using dendritic cells loaded with an allogeneic tumor cell lysate Cancer Immunol Immunother 2005 Sep 27: 1-11 [In electronic edition before printed]).

To this end, mature dendritic cells that have been matured in vitro from immature dendritic cells from a patient are loaded with tumor-specific antigens and injected back into the body, preferably in the lymph nodes or in their proximity . Inside the lymph nodes the dendritic cells interact with the natural T lymphocytes resulting in the transduction of active signals during the so-called immunological synapse and the subsequent proliferation of effector T lymphocytes, which in turn mediate antitumor responses such as cytotoxicity ( cytotoxic T lymphocytes = CTL), macrophage activation and delayed type hypersensitivity reactions. DCs regulate the polarization of auxiliary T lymphocytes (h) CD4 +. Th1 lymphocytes, for example, maintain CTLs by secretion of certain cytokine patterns (for example, interferon gamma and IL-2, TNF-beta). On the other hand, Th2 lymphocytes induce antibodies as well as eosinophils and mast cell degranulation by IL-4, IL-5, IL-10 and IL-13 (Langenkamp A, Messi M, Lanzavecchia A, Sallusto F. Kinetics of dendritic cell activation: impact on priming of Th1, Th2 and nonpolarized T cells. Nat. Immunol. 2000 Oct; 1 (4): 311-6, O'Gara, A: Cytokines induces the development of functionally heterogeneous T helper cell subsets. Immunity 1998, 8: 275-283, Rengarajan J, Szabo SJ, Glimcher LH. Transcriptional regulation of Th1 / Th2 polarization. Immunol. Today. 2000 Oct; 21 (10): 479-83. Review).

For dendritic cell therapy, it is essential that a sufficient number of major CDs be available. Since, in a patient, only 0.2% of leukocytes are dendritic cells, it is necessary to have an effective method for in vitro production of mature dendritic cells.

Various methods have been proposed in the art for the preparation of mature dendritic cells from peripheral blood mononuclear cells, monocytes or other myeloid progenitor cells (Jonuleit H, Kuhn U, Muller G, Steinbrink K, Paragnik L, Schmitt E, Knop J, Enk AH .: Pro-inflammatory cytokines and prostaglandins induce maturation of potent immunostimulatory dendritic cells under fetal calf serum-free conditions Eur. J. Immunol. 1997 Dec; 27 (12): 3135-42). PJ fly, Hobeika AC, Clay TM, Nair SK, Thomas EK, Morse MA, Lyerly HK. A subset of human monocyte-derived dendritic cells expresses high levels of interleukin-12 in response to combined CD40 ligand and interferon-gamma treatment. Blood. 2000 Nov 15; 96 (10): 3499-504. Mailliard RB, Wankowicz-Kalinska A, Cai Q, Wesa A, Hilkens CM, Kapsenberg ML, Kirkwood JM, Storkus WJ, Kalinski P .: Alpha-type-1 polarized dendritic cells: a novel immunization tool with optimized CTL-inducing activity. Cancer Res. 2004 Sep 1; 64 (17): 5934-7. Napolitani G, Rinaldi A, Bertoni F, Sallusto F, Lanzavecchia A .: Selected Toll-like receptor agonist combinations synergistically trigger a T helper type 1-polarizing program in dendritic cells. Nat. Immunol. 2005 Aug; 6 (8): 769-76. Epub 2005 Jul 3. 2005 Aug; 6 (8): 769-76. 2005. Gautier G, Humbert M, Deauvieau F, Scuiller M, Hiscott J, Bates EE, Trinchieri G, Caux C, Garrone P .: A type I interferon autocrine-paracrine loop is involved in Toll-like receptor-induced interleukin-12p70 secretion by dendritic cells. J. Exp. Med. 2005 May 2; 201 (9): 1435-46. 2005).

It is accepted that the culture of peripheral blood mononuclear cells, monocytes or other myeloid progenitor cells with GM-CSF and with IL-4 or IL-13 results in the in vitro production of immature dendritic cells (Ahn JS, Agrawal B. IL -4 is more effective than IL-13 for in vitro differentiation of dendritic cells from peripheral blood mononuclear cells. Int. Immunol. 2005 Oct; 17 (10): 1337-46. Epub 2005 Sep 2). However, to date, there is no satisfactory method available for maturation of immature dendritic cells. Jonuleit H. et al. (1997, supra) describe a ripening process using a composition comprising TNF-α, IL-1, IL-6 and prostaglandin E2 (PG) (the so-called Jonuleit cocktail). Dendritic cells produced by incubation of immature dendritic cells with this composition show surface markers of mature dendritic cells and can thus be collected. However, these cells do not produce biological active IL-12p70, which is the most important factor for the induction of Th1 cells in the lymph nodes.

Mailliard, R. et al., Describe a composition comprising TNF-α, IL-1, interferon α, interferon γ and poly (I: C) (Mailliard, R. et al., 2004, supra.). In contrast to the previous Jonuleit cocktail, incubation of immature dendritic cells with this cocktail called Kalinski results in mature dendritic cells (as demonstrated by the respective surface markers), which produce IL-12p70. However, these cells are very

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adherent to the bottom of the culture flasks and are, therefore, almost impossible to collect. It is, therefore, very difficult, if not impossible, to obtain with this method mature dendritic cells sufficient for vaccination therapy.

WO 00/47719 describes a compound (R848) that is proposed for the preparation of mature dendritic cells. In the experiments described in this application, immature dendritic cells are stimulated only with R848. However, R848 as the only maturation substance is not able to provide all the appropriate characteristics for clinical dendritic cells. All experiments have been carried out with fetal calf serum (FCS) and, therefore, not applicable under conditions of good manufacturing practice (GMP) because fetal calf-free conditions are crucial to a process. from GMP.

Therefore, improved methods for the preparation of mature dendritic cells from immature dendritic cells are still needed.

The invention provides a method for in vitro maturation of at least one immature dendritic cell, which comprises stimulating said immature dendritic cell with TNF-α, IL-1β, IFNγ, a TLR7 / 8 agonist and prostaglandin E2 (PG).

The present invention is based on the surprising finding that the combination of TNF-α, IL-1β, IFNγ, a TLR7 / 8 agonist and prostaglandin E2 (PG) is especially suitable for promoting in vitro maturation of dendritic cells. Especially, and as demonstrated in the Example, mature dendritic cells obtained using said combination surprisingly express IL-12p70 in considerable amounts and are surprisingly easy to collect, allowing mature dendritic cells to be obtained in considerable quantities. Such mature dendritic cell populations could not be produced with the cocktails known in the art, and especially not with the Jonuleit cocktail or with the Kalinski cocktail, as explained above.

Individual methods for the preparation of mature dendritic cells, for example, from human peripheral blood mononuclear cells, monocytes or other myeloid progenitor cells, and from the same immature DCs, which have been isolated directly from the blood, are known. in the art (Berger TG, Strasser E, Smith R, Carste C, Schuler-Thurner B, Kaempgen E, Schuler G. Efficient elutriation of monocytes within a closed system (Elutra) for clinical-scale generation of dendritic cells. J. Immunol Methods. 2005 Mar; 298 (1-2): 61-72. Errata in: J. Immunol. Methods. 2005 Aug; 303 (1-2): 152. Strasser EF, Berger TG, Weisbach V, Zimmermann R, Ringwald J, Schuler-Thurner B, Zingsem J, Eckstein R. Comparison of two apheresis systems for the collection of CD14 + cells intended to be used in dendritic cell culture. Transfusion. 2003 Sep; 43 (9): 1309-16. : Transfusion. 2003 Oct; 43 (10): 1502. Campbell JD, Piechaczek C, Winkels G, Schwamborn E, Micheli D, He nnemann S, Schmitz J. Isolation and generation of clinical-grade dendritic cells using the CliniMACS system. Methods Mol. Med. 2005; 109: 55-70. Dubsky P, Ueno H, Piqueras B, Connolly J, Banchereau J, Palucka AK. Human dendritic cell subsets for vaccination. J. Clin. Immunol 2005 Nov; 25 (6): 551-72).

Therefore, basic methods, such as incubation periods, means used, etc., to produce mature dendritic cells from immature dendritic cells, are known in the art. The present invention relates to the use of a new combination of factors in the context of these prior art methods. The method of the present invention can, therefore, be easily performed by those skilled in the art, simply by performing the prior art methods, but using the combination of factors identified above during the incubation of immature dendritic cells in order to get mature dendritic cells.

In addition, since each of the individual components has already been used individually in the art, those skilled in this art can easily determine to what concentration each factor should be used. Additionally, the expert would be able to adapt the individual concentration of a given factor depending on the compositions of serum factors and components that grow especially in the cell culture medium.

As a general guideline, TNF-α and IL-1β could be used at concentrations from 1 ng / mL to 50 ng / mL, more preferably from 5 ng / mL to 40 ng / mL, and even more preferably at 10 ng / mL. PG could be used at concentrations from 50 ng / mL to 5000 ng / mL, preferably from 50 ng / mL to 1000 ng / mL, even more preferably from 50 ng / mL to 500 ng / mL or at concentrations of 100 ng / mL or 250 ng / mL. The IFNγ could be used at a concentration between 500 U / mL and 10,000 U / mL, preferably between 1000 and 5000 U / mL, and more preferably at 1000 or 5000 U / mL. Finally, the TLR7 / 8 agonist, preferably R848, could be used at a concentration between 0.2 and 5 µg / mL, preferably 0.5 µg / mL to 2 µg / mL, more preferably 1 µg / mL.

According to the invention, immature dendritic cells are cultured with the above combination of factors. This can be done by adding the factors to the culture medium. Alternatively, the culture medium in which the immature dendritic cells have been cultured is replaced by a medium that already contains the factors. In a more preferred embodiment, the aforementioned substances are part of a composition added to the culture medium of said immature dendritic cell.

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Said culture medium may be of any suitable type, that is, it may or may not contain human serum, it may or may not be supplemented with other animal supplements, such as proteins, amino acids or antibiotics. In a preferred embodiment, the medium is produced and used under conditions of good manufacturing practice (GMP).

After the maturation period is complete, DC can be collected by pipetting, agitation (manual or mechanical) and washing with saline solution, media components (e.g. RPMI) or complete medium without cytokines. The cells can be collected, centrifuged and the cytokines can be washed away by performing at least one or more new suspensions of the settled DCs.

Immature dendritic cells can be further treated with a TLR3 ligand, preferably poly (I: C), for example, at a concentration between 10 and 50 ng / mL, preferably 20 ng / mL. The TLR3 ligand can be added separately to the cells or it can be part of the composition that also comprises the other factors.

In a preferred embodiment of the invention, said TLR7 / 8 agonist is an immune response modifying imidazoquinoline compound, more preferably 4-amino-2-ethoxymethyl-α, α-dimethyl-1 H -imidazole [4,5c] quinoline-1-ethanol (R848). The production of said compound is described in detail in WO 00/47719. However, other TLR7 / 8 agonists such as imiquimod (R837), guanine analogue loxoribin, TLR8 agonists such as single stranded RNAs that bind TLR7 / 8, for example, single stranded poly (U) and single stranded RNA40 can also be used. or combinations of TLR7 / 8 agonists.

In a more preferred embodiment, the immature dendritic cell used as a starting material of the method of the invention is an immature dendritic cell derived from monocytes. Preferably, monocytic progenitors obtained from peripheral blood or by leukopheresis and enriched by density gradient centrifugation, elutriation or simply plastic adhesion techniques are used.

Alternatively, it is also possible to obtain monocytic progenitor cells from CD34 + progenitor cells by in vitro differentiation to CD14 + cells, for example, with FLT3L, SCF, TPO, IL-3 and / or IL-6.

Preferably, said immature dendritic cell is obtained by incubating peripheral blood mononuclear cells, monocytes or other human myeloid progenitor cells with GM-CSF and IL-4 or IL-13. As already stated above, the corresponding methods are known in the art. In addition, said methods are described in the Example.

Any suitable means for physiological conditioning of mammalian cells can be used, for example, containing standard amino acids, growth factors, carbon source, buffer system or certain salts. Cell culture can be performed at 37 ° C according to the composition of the medium at certain concentrations of CO2.

In addition, immature DCs can be obtained directly from peripheral blood, for example, by leukopheresis.

In an especially preferred embodiment, the immature dendritic cell is of human origin, although situations may arise, for example, applications in scientific research or veterinary medicine, in which immature dendritic cells whose origin is from another mammal can be used.

Accordingly, in a further preferred embodiment, the method of the invention comprises the following steps:

a) prepare mononuclear cells from peripheral blood,

b) incubate the mononuclear cells of step a) with GM-CSF and IL-4 or IL-13,

c) incubating the cells obtained in step b) with a cocktail comprising TNFα, IL-1β, IFNγ, a TLR7 / 8 agonist, prostaglandin E2 (PG) and, optionally, a TLR3 agonist, preferably poly (I: C), and

d) collect the mature dendritic cell or cells.

In stage a), mononuclear cells can be obtained by peripheral blood leukopheresis. In addition, mononuclear cells can be isolated by magnetic separation or by FACS, elutriation or adhesion to plastic or density gradient centrifugation (eg, metricamide)

Preferably, the incubation in step b) lasts from 1 to 9, preferably from 2 to 9, more preferably from 2 to 6 days. However, it is also feasible to suppress steps a) or b) if immature DCs newly isolated from peripheral blood / by leukopheresis are used. In addition, it is possible that stage b) lasts only hours and can be performed in combination with stage c).

The incubation in step c) can last from 12 hours to 72 hours, preferably 24 hours or 20 hours.

As discussed above, those skilled in the art may adapt these incubation periods, if necessary.

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Incubation of immature dendritic cells and collection have already been described above.

In a more preferred embodiment of the invention, the mature dendritic cell or cells are further loaded in vitro with one or more antigens. The loading of mature cells with said antigens is described in more detail below.

Preferably, it is assumed that said antigen or antigens cause maturation of effector T lymphocytes within the lymph nodes.

More preferably, and as also described below, said loading is performed by incubating the mature dendritic cell or cells with at least one peptide of said antigen or transfecting the dendritic cell or cells with RNA or DNA encoding the antigen.

The invention further relates to a population of mature dendritic cells, obtainable by the method of the invention, wherein the immature dendritic cell is of human origin, and where TNFα is used at a concentration of 1 ng / mL at 50 ng / mL, IL -1β is used at a concentration of 1 ng / mL at 50 ng / mL, IFNγ is used at a concentration of 500 U / mL at 10,000 U / mL, the TLR7 / 8 agonist is used at a concentration of 0.2 µg / mL at 5 µg / mL, PGE2 is used at a concentration of 50 ng / mL at 5000 ng / mL and the TLR3 agonist is used at a concentration of 10 ng / mL at 50 ng / mL.

As discussed above, mature dendritic cells obtained by the method of the invention produce considerable amounts of IL-12p70 and are easy to collect. These combined effects were not observed with either Jonuleit or Kalinski's cocktail in the experiments presented herein (see Example).

As demonstrated in the Example and especially in Figures 7 and 8, the population of mature dendritic cells of the invention is capable of stimulating the production of interferon-gamma from both natural killer cells (Figure 8) and specific T lymphocytes. of peptides (Figure 7).

Consequently, the dendritic cells obtainable by the method of the invention are especially suitable in the context of in vivo activation of T lymphocytes, in order to treat diseases in which such activation of T lymphocytes is necessary. Consequently, in a In addition, the present invention also relates to a pharmaceutical composition comprising the population of mature dendritic cells. In addition, the invention also relates to the population of mature dendritic cells for use in the treatment of a disease selected from the group consisting of oncogenic diseases and infectious diseases (for example, caused by viruses, bacteria, bacteria or intracellular fungi).

In a preferred embodiment, said population of mature dendritic cells is obtainable by a method of the invention in which the cells are also incubated with poly (I: C). As indicated above, said dendritic cells are especially capable of stimulating NK cells and are as potent as cells incubated according to the invention without poly (I: C) in stimulating peptide-specific T lymphocytes.

Preferably, for the treatment of the above diseases, dendritic cells are loaded in vitro with antigens that are supposed to cause maturation of effector T lymphocytes within the lymph nodes. Such methods are known in the art (Dieckmann D, Schultz ES, Ring B, Chames P, Held G, Hoogenboom HR, Schuler G. Optimizing the exogenous antigen loading of monocyte-derived dendritic cells. Int. Immunol. 2005 May; 17 ( 5): 621-35 Epub 2005 Apr 11. Kikuchi T, Akasaki Y, Abe T, Fukuda T, Saotome H, Ryan JL, Kufe DW, Ohno

T. Vaccination of glioma patients with fusions of dendritic and glioma cells and recombinant human interleukin-12. J. Immunother. 2004 Nov-Dec; 27 (6): 452-9. Gong J, Koido S, Kato Y, Tanaka Y, Chen D, Jonas A, Galinsky I, DeAngelo D, Avigan D, Kufe D, Stone R. Induction of anti-leukemic cytotoxic T lymphocytes by fusion of patientderived dendritic cells with autologous myeloblasts . Leuk Res. 2004 Dec; 28 (12): 303-12. Grunebach F, Kayser K, Weck MM, Muller MR, Appel S, Brossart P. Cotransfection of dendritic cells with RNA coding for HER-2 / neu and 4-1 BBL increases the induction of tumor antigen specific cytotoxic T lymphocytes. Cancer Gene Ther. 2005 Sep; 12 (9): 749-56. Kyte JA, Kvalheim G, Aamdal S, Saeboe-Larssen S, Gaudernack G. Preclinical full-scale evaluation of dendritic cells transfected with autologous tumor-mRNA for melanoma vaccination. Cancer Gene Ther. 2005 Jun; 12 (6): 579-91. Navabi H, Croston D, Hobot J, Clayton A, Zitvogel L, Jasani B, Bailey-Wood R, Wilson K, Tabi Z, Mason MD, Adams M. Preparation of human ovarian cancer ascites-derived exosomes for a clinical trial. Blood Cells Mol. Dis. 2005 Sep-Oct; 35 (2): 149-52. Escudier B, Dorval T, Chaput N, Andre F, Caby MP, Novault S, Flament C, Leboulaire C, Borg C, Amigorena S, Boccaccio C, Bonnerot C, Dhellin O, Movassagh M, Piperno S, Robert C, Serra V , Valente N, Le Pecq JB, Spatz A, Lantz O, Tursz T, Angevin E, Zitvogel L. Vaccination of metastatic melanoma patients with autologous dendritic cell (DC) derived-exosomes: results of the first phase I clinical trial. J. Transl. Med. 2005 Mar 2; 3 (1): 10. Kawamura K, Kadowaki N, Suzuki R, Udagawa S, Kasaoka S, Utoguchi N, Kitawaki T, Sugimoto N, Okada N, Maruyama K, Uchiyama T. Dendritic cells that endocytosed antigen-containing IgG-liposomes elicit effective antitumor immunity. J. Immunother. 2006 Mar-Apr; 29 (2): 165-74. Griffioen M, Borghi M, Schrier PI, Osanto S, Schadendorf D. Analysis of T-cell responses in metastatic melanoma patients vaccinated with dendritic cells pulsed with tumor lysates. Immunol cancer. Immunother 2004 Aug; 53 (8): 715-22. Epub 2004 Mar 3. Su Z, Dannull J, Yang BK, Dahm P, Coleman D, Yancey D, Sichi S, Niedzwiecki D, Boczkowski D, Gilboa E, Vieweg J.

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Telomerase mRNA-transfected dendritic cells stimulate antigen-specific CD8 + and CD4 + T cell responses in patients with metastatic prostate cancer. J. Immunol. 2005 Mar 15; 174 (6): 3798-807).

The loading of dendritic cells with respective antigens could be performed by competitive displacement of peptides into solutions of the MHC-binding groove, or for more complex antigens, such as original tumor lysates and proteins or lysates of tumor cell lines, by phagocytosis of immature DC and appropriate treatment. Methods of transfection (lipofection, electroporation or simply incubation of naked nucleic acids) and the introduction of nucleic acids, such as plasmids encoding antigens, RNA or DNA thereof, and especially RNA from original tumors or tumor cell lines in the cells are also feasible. DC It may also be possible to use other antigenic combinations with original MHC molecules, such as membrane fragments or exosomes, as sources of antigens of any kind.

As indicated above, the population of mature dendritic cells can be administered directly to the body to produce active T lymphocytes against, for example, a selected type of cancer cell. The administration of these cells, often with pharmaceutically acceptable carriers, is carried out by any of the routes normally used for the introduction of a cell in final contact with the blood cells or mammalian tissues.

Formulations suitable for parenteral administration, such as, for example, intra-articularly (in the joints), intravenously, intramuscularly, intradermally, intraperitoneally and subcutaneously (preferably intradermally or subcutaneously), include vehicles such as isotonic sterile injection solutions aqueous, which may contain antioxidants, buffers, bacteriostatic and solute agents that make the formulation isotonic with the intended recipient's blood, and sterile aqueous and non-aqueous suspensions that may include suspending agents, solubilizers, thickeners, stabilizers and preservatives Intravenous or intraperitoneal administration is the preferred method of administration for dendritic cells of the invention.

The dose of dendritic cells administered to a patient, in the context of the present invention, must be sufficient to produce a beneficial therapeutic response over time to the patient, or to inhibit the growth of cancer cells or to inhibit an infection. Thus, the cells are administered to a patient in an amount sufficient to elicit an effective CTL response to the virus or tumor antigen and / or to alleviate, reduce, cure or at least partially stop the symptoms and / or complications of the disease or infection. An adequate amount to achieve this is defined as a "therapeutically effective dose." The dose will be determined taking into account the activity of the dendritic cells produced and the condition of the patient, as well as the weight or surface area of the patient to be treated. The magnitude of the dose will also be determined taking into account the existence, nature and extent of any adverse side effects that accompany the administration of a particular cell in a particular patient. To determine the effective amount of the cell to be administered in the treatment or prophylaxis of diseases such as cancer (for example, metastatic melanoma, prostate cancer, etc.), the doctor needs to assess circulating plasma levels, toxicity of the CTL, the progression of the disease and the induction of the immune response against any type of cell introduced.

Before the infusion, blood samples are obtained and preserved for analysis. Generally, at least about 104 to 106, and typically between 108 and 1010, cells are infused intravenously or intraperitoneally to a 70 kg patient for about 60-120 minutes. Preferably, cell numbers of at least 107 / vaccination point are used. Injections may be repeated, for example 4 times, in a 2 week interval and should preferably be administered near the lymph nodes by intradermal or subcutaneous injections. Souvenir injections can be done after a 4-week break. Vital signs and oxygen saturation by pulse oximetry are carefully monitored. Blood samples are obtained 5 minutes and 1 hour after the infusion and are preserved for analysis. Approximately every month the reinfusion of cells is repeated in a total of 10-12 treatments in a period of one year. After the first treatment, infusions can be performed in the outpatient clinic according to the doctor's criteria. If reinfusion is administered on an outpatient basis, the participant is monitored for at least 4 hours after each therapy.

As for its administration, the cells of the present invention can be administered at a rate determined by the LD50 (or other measure of toxicity) of the cell type and the side effects of the cell type at various concentrations, relative to the weight and the general health of the patient. Administration can be done in a single dose or in divided doses. The cells of this invention can complement other treatments of a state by known conventional therapy, including cytotoxic agents, nucleotide analogs and biological response modifiers. Similarly, optionally add modifiers of the biological response to dendritic cell treatment. For example, the cells are optionally administered with an adjuvant, a cytokine, such as GM-CSF, IL-12 or IL-2, or with KLH.

As indicated above, the invention also relates to the combined use of TNF-α, IL-1β, IFNγ, a TLR7 / 8 agonist, prostaglandin E2 (PG) and, optionally, a TLR3 agonist, preferably poly (I : C) for the preparation of at least one mature dendritic cell. In addition, the invention also relates to a composition comprising TNF-α, IL-1β, IFNγ, a TLR7 / 8 agonist, prostaglandin E2 (PG) and, optionally, a TLR3 agonist, preferably poly (I: C) . As indicated above, in both cases,

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preferably said TLR7 / 8 agonist is an imidazoquinoline-type immune response modifying compound, preferably 4-amino-2-ethoxymethyl-α, α-dimethyl-1H-imidazol [4,5-c] quinoline-1-ethanol ( R848).

Brief Description of the Figures

Figure 1:

Generation of different matured dendritic cells (DC)

A. Recovery of DC collected after primary cell culture (6 days of differentiation + 24 hours of maturation) calculated in relation to all seeded cells (mononuclear cells) or CD14 + monocytes detected by manual counting in the Neubauer chamber and FACS analysis (CD14). The viability detected due to the quantitative incorporation of 7AAD was measured within FL-3 of FACS Calibur. The dashed line indicates the levels of DC matured with the gold standard Jonuleit cocktail.

B. Surface expression of specific DC molecules for DCs after primary DC culture. The low expression of CD14 compared to the high expression of the CD83 specific molecule of DC indicates a mature state of the DC, detected by FACS analysis (percentage of all cells without restriction window, acquisition of 10,000 cells in total).

C. Surface expression of the co-stimulatory molecules, CD80 and CD86, after primary DC culture detected by FACS analysis. Chemokine receptor 7 expression (CCR7 = CD197) as an indication of the migratory potential of DCs in the lymph nodes. A positive percentage is detected according to the superposition with the isotypic control antibody.

DC1 = Jonuleit = TNF-alpha (10 ng / mL) + IL-1 beta (10 ng / mL) + IL-6 (15 ng / mL) + Prostaglandin E2 (= PGE2, 1000 ng / mL)

DC2 = Kalinski = TNF-alpha (10 ng / mL) + IL-1 beta (10 ng / mL) + IFN alpha (3000 IU / mL) + IFN gamma (1000 IU / mL) + poly (I: C) (20 ng / mL)

DC3 = TNF-alpha (10 ng / mL) + IL-1 beta (10 ng / mL) + PGE2 (100 ng / mL) + IFN gamma (1000 IU / mL) + R848 (1 µg / mL)

DC4 = TNF-alpha (10 ng / mL) + IL-1 beta (10 ng / mL) + PGE2 (250 ng / mL) + IFN gamma (5000 IU / mL) + R848 (1 µg / mL)

DC5 = TNF-alpha (10 ng / mL) + IL-1 beta (10 ng / mL) + PGE2 (250 ng / mL) + IFN gamma (5000 IU / mL) + R848 (1 µg / mL) + poly (I : C) (20 ng / mL)

Figure 2:

Stability of maturation (washing separation test) of different matured DCs

The DCs were washed away from the cytokines and grown 40 hours after maturation with a DC culture medium only with serum and gentamicin.

A. Viability of different DC matured after washing separation detected due to the incorporation of 7AAD.

B. Low levels of surface expression of induced CD14 compared to high levels of CD83 expression after separation by washing.

C. Expression of the co-stimulatory molecules, CD80 and CD86, and CCR7 after separation by washing.

DC1 = Jonuleit = TNF-alpha (10 ng / mL) + IL-1 beta (10 ng / mL) + IL-6 (15 ng / mL) + Prostaglandin E2 (= PGE2, 1000 ng / mL)

DC2 = Kalinski = TNF-alpha (10 ng / mL) + IL-1 beta (10 ng / mL) + IFN alpha (3000 IU / mL) + IFN gamma (1000 IU / mL) + poly (I: C) (20 ng / mL)

DC3 = TNF-alpha (10 ng / mL) + IL-1 beta (10 ng / mL) + PGE2 (100 ng / mL) + IFN gamma (1000 IU / mL) + R848 (1 µg / mL)

DC4 = TNF-alpha (10 ng / mL) + IL-1 beta (10 ng / mL) + PGE2 (250 ng / mL) + IFN gamma (5000 IU / mL) + R848 (1 µg / mL)

DC5 = TNF-alpha (10 ng / mL) + IL-1 beta (10 ng / mL) + PGE2 (250 ng / mL) + IFN gamma (5000 IU / mL) + R848 (1 µg / mL) + poly (I : C) (20 ng / mL)

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Figure 3:

Cryopreservation of different burnings.

DCs were frozen and preserved under a gaseous phase of liquid nitrogen and analyzed after thawing.

A. Viability of different DC matured after washing separation detected due to the incorporation of 7AAD.

B. Low levels of CD14 surface expression compared to high levels of CD83 expression after freezing.

C. Expression of co-stimulatory molecules, CD80 and CD86, and CCR7 after freezing.

DC1 = Jonuleit = TNF-alpha (10 ng / mL) + IL-1 beta (10 ng / mL) + IL-6 (15 ng / mL) + Prostaglandin E2 (= PGE2, 1000 ng / mL)

DC2 = Kalinski = TNF-alpha (10 ng / mL) + IL-1 beta (10 ng / mL) + IFN alpha (3000 IU / mL) + IFN gamma (1000 IU / mL) + poly (I: C) (20 ng / mL)

DC3 = TNF-alpha (10 ng / mL) + IL-1 beta (10 ng / mL) + PGE2 (100 ng / mL) + IFN gamma (1000 IU / mL) + R848 (1 µg / mL)

DC4 = TNF-alpha (10 ng / mL) + IL-1 beta (10 ng / mL) + PGE2 (250 ng / mL) + IFN gamma (5000 IU / mL) + R848 (1 µg / mL)

DC5 = TNF-alpha (10 ng / mL) + IL-1 beta (10 ng / mL) + PGE2 (250 ng / mL) + IFN gamma (5000 IU / mL) + R848 (1 µg / mL) + poly (I : C) (20 ng / mL)

Figure 4:

Analysis of the allo-stimulatory capacity of different DCs matured in a mixed lymphocyte reaction.

A. Functional control of the proliferating capacity of autologous T lymphocytes: only T lymphocytes within the medium, T lymphocytes stimulated with the third part (= five mixed MNC donors) (irradiated 105 / well with 4000 rad, stimulant lymphocyte ratio: lymphocytes 1: 1 responders), T + IL-2 lymphocytes (5 IU / mL) and PHA 10 μg / mL last 68 hours, T lymphocytes stimulated with 50 IU / mL of IL-2. Number of T 105 lymphocytes / well. After a co-culture for 7 days, proliferation was measured by incorporation of 3H-thymidine the last 24 hours. All values are calculated from five repeated wells.

B. Functional control of the proliferative capacity of an illustrative allogeneic responding T lymphocyte.

C. Negative control of proliferation of different irradiated matured DCs (4000 rad) (104 / well, according to the number of lymphocytes in the DC test ratio: 1:10 responding lymphocytes)

D. Proliferation of autologous T lymphocytes stimulated by different matured DCs (number of DC 104 / well, number of T lymphocytes 105, ratio of DC: 1:10 responding lymphocytes)

E. Proliferation of an illustrative allogeneic responding T lymphocyte stimulated by different matured DCs (number of DC 104 / well, number of T lymphocytes 105, DC ratio: 1:10 responding lymphocytes).

F. Summary of proliferation data of three independent responding T lymphocytes compared to autologous T lymphocytes stimulated by different matured DC.

DC1 = Jonuleit = TNF-alpha (10 ng / mL) + IL-1 beta (10 ng / mL) + IL-6 (15 ng / mL) + Prostaglandin E2 (= PGE2, 1000 ng / mL)

DC2 = Kalinski = TNF-alpha (10 ng / mL) + IL-1 beta (10 ng / mL) + IFN alpha (3000 IU / mL) + IFN gamma (1000 IU / mL) + poly (I: C) (20 ng / mL)

DC3 = TNF-alpha (10 ng / mL) + IL-1 beta (10 ng / mL) + PGE2 (100 ng / mL) + IFN gamma (1000 IU / mL) + R848 (1 µg / mL)

DC4 = TNF-alpha (10 ng / mL) + IL-1 beta (10 ng / mL) + PGE2 (250 ng / mL) + IFN gamma (5000 IU / mL) + R848 (1 µg / mL)

DC5 = TNF-alpha (10 ng / mL) + IL-1 beta (10 ng / mL) + PGE2 (250 ng / mL) + IFN gamma (5000 IU / mL) + R848 (1 µg / mL) + poly (I : C) (20 ng / mL)

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Figure 5:

Production of IL-12p70 and IL-10 by DC matured using different cocktails

Immature DC were grown with different ripening cocktails and the amounts of IL-12p70 and IL-10 were determined by standard ELISA. The filled bars indicate IL-12p70 and the empty bars IL-10, respectively.

A. Supernatant medium of primary maturation cultures after 7 days.

B. Supernatant medium from mature and washed DC cultures and fibroblasts transfected by CD40L after a 24-hour co-culture period, which represents a signal 3 assay as described in Materials and Methods.

C. IL-12p70 / IL-10 ratios were determined for matured DC populations in different cocktails, based on the pg / mL values of the signal 3 assay.

For the calculation it was assumed that IL-12p70 and IL-10 have a theoretically equal biological potential. The filled circles indicate a positive ratio between 0 and 3.5, the dotted lines assess the intense differences of the DC matured with the Jonuleit or Kalinski cocktail for the secretion of IL-12p70.

DC1 = Jonuleit = TNF-alpha (10 ng / mL) + IL-1 beta (10 ng / mL) + IL-6 (15 ng / mL) + Prostaglandin E2 (= PGE2, 1000 ng / mL)

DC2 = Kalinski = TNF-alpha (10 ng / mL) + IL-1 beta (10 ng / mL) + IFN alpha (3000 IU / mL) + IFN gamma (1000 IU / mL) + poly (I: C) (20 ng / mL)

DC3 = TNF-alpha (10 ng / mL) + IL-1 beta (10 ng / mL) + PGE2 (100 ng / mL) + IFN gamma (1000 IU / mL) + R848 (1 µg / mL)

DC4 = TNF-alpha (10 ng / mL) + IL-1 beta (10 ng / mL) + PGE2 (250 ng / mL) + IFN gamma (5000 IU / mL) + R848 (1 µg / mL)

DC5 = TNF-alpha (10 ng / mL) + IL-1 beta (10 ng / mL) + PGE2 (250 ng / mL) + IFN gamma (5000 IU / mL) + R848 (1 µg / mL) + poly (I : C) (20 ng / mL)

Figure 6:

Expression of EGFP in DC transfected with in vitro transcribed RNA encoding EGFP

Overlays of flow cytometry histograms show the transfected RNA of the EGFP in mature DC (filled curves) 24 hours after electroporation and the corresponding untransfected DC (empty curves) as negative controls. The DCs were matured in the four indicated cocktails, the RNA was introduced by electroporation, the DCs were returned to their corresponding media containing the ripening cocktails and were collected for flow cytometry 24 hours later. The numbers indicate the percentages of EGFP positive DCs and their average fluorescence intensities. These data are representative of two experiments with measurements at 24 and 48 hours.

DC1 = Jonuleit = TNF-alpha (10 ng / mL) + IL-1 beta (10 ng / mL) + IL-6 (15 ng / mL) + Prostaglandin E2 (= PGE2, 1000 ng / mL)

DC2 = Kalinski = TNF-alpha (10 ng / mL) + IL-1 beta (10 ng / mL) + IFN alpha (3000 IU / mL) + IFN gamma (1000 IU / mL) + poly (I: C) (20 ng / mL)

DC4 = TNF-alpha (10 ng / mL) + IL-1 beta (10 ng / mL) + PGE2 (250 ng / mL) + IFN gamma (5000 IU / mL) + R848 (1 µg / mL)

DC5 = TNF-alpha (10 ng / mL) + IL-1 beta (10 ng / mL) + PGE2 (250 ng / mL) + IFN gamma (5000 IU / mL) + R848 (1 µg / mL) + poly (I : C) (20 ng / mL)

Figure 7:

Autologous lymphocyte response of a donor positive to HLA-A * 0201 to DC pulsed with virus peptides

T lymphocyte responses were evaluated in an IFNγ-ELISPOT experiment using lymphocytes (fraction 3 of Elutra enriched with T lymphocytes = 54.76% of CD3 + cells) that were first activated for 7 days with mature DC pulsed with peptides already then stimulated again for 24 hours with monocytes plus CEF peptides. For ELISPOT analyzes, 4x103 autologous in vitro activated lymphocytes were stimulated with 2 x 103 monocytes together with the mixture of five CEF peptides. Mean ± standard error for wells was calculated by

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triplicate. Note: Due to insufficient recoveries, lymphocytes activated by DC2 cells were not included in the assay.

DC1 = Jonuleit = TNF-alpha (10 ng / mL) + IL-1 beta (10 ng / mL) + IL-6 (15 ng / mL) + Prostaglandin E2 (= PGE2, 1000 ng / mL)

DC2 = Kalinski = TNF-alpha (10 ng / mL) + IL-1 beta (10 ng / mL) + IFN alpha (3000 IU / mL) + IFN gamma (1000 IU / mL) + poly (I: C) (20 ng / mL)

DC3 = TNF-alpha (10 ng / mL) + IL-1 beta (10 ng / mL) + PGE2 (100 ng / mL) + IFN gamma (1000 IU / mL) + R848 (1 µg / mL)

DC4 = TNF-alpha (10 ng / mL) + IL-1 beta (10 ng / mL) + PGE2 (250 ng / mL) + IFN gamma (5000 IU / mL) + R848 (1 µg / mL)

DC5 = TNF-alpha (10 ng / mL) + IL-1 beta (10 ng / mL) + PGE2 (250 ng / mL) + IFN gamma (5000 IU / mL) + R848 (1 µg / mL) + poly (I : C) (20 ng / mL)

Figure 8:

Interferon-gamma response of autologous lymphocytes stimulated with mature DC

Lymphocytes from a donor with HLA-A * 0201 with autologous DC matured were stimulated using the Jonuleit cocktail, the Kalinski cocktail and the three new cocktails described herein.

Peripheral blood lymphocytes (PBL) underwent elutriation and the contents in fraction 3 were separated, in which 54.8% of the cells were CD3 + T lymphocytes and 17.7% CD56 + cells, which is characteristic of natural killer cells (NK). (A) The PBLs of fraction 3 of elutriation were incubated directly with the different matured DC populations using the different cocktails and their interferon-gamma secretion (IFNgamma) was measured after 24 hours in a standard ELISPOT assay.

DC1 = Jonuleit = TNF-alpha (10 ng / mL) + IL-1 beta (10 ng / mL) + IL-6 (15 ng / mL) + Prostaglandin E2 (= PGE2, 1000 ng / mL)

DC2 = Kalinski = TNF-alpha (10 ng / mL) + IL-1 beta (10 ng / mL) + IFN alpha (3000 IU / mL) + IFN gamma (1000 IU / mL) + poly (I: C) (20 ng / mL)

DC3 = TNF-alpha (10 ng / mL) + IL-1 beta (10 ng / mL) + PGE2 (100 ng / mL) + IFN gamma (1000 IU / mL) + R848 (1 µg / mL)

DC4 = TNF-alpha (10 ng / mL) + IL-1 beta (10 ng / mL) + PGE2 (250 ng / mL) + IFN gamma (5000 IU / mL) + R848 (1 µg / mL)

DC5 = TNF-alpha (10 ng / mL) + IL-1 beta (10 ng / mL) + PGE2 (250 ng / mL) + IFN gamma (5000 IU / mL) + R848 (1 µg / mL) + poly (I : C) (20 ng / mL).

Example

The following example represents the description of an experiment as a representative example of at least three independent experiments performed using cells from different donors.

1. Material and methods

Leukopheresis and elutriation

To obtain monocytes as a population of progenitor cells for the generation of human dendritic cells, the inventors used a closed ELUTRA elutriation system (Gambro BCT, Lakewood, USA).

After informed consent, healthy, immobilized donors underwent leukopheresis for 180 minutes with the COBE Spectra cell separator (Gambro BCT, Inc. Lakewood, USA) using a modified MNC program (V6.1): the separation factor was adjusted to 700 with a collection rate of 0.8 mL / min and a target hematocrit of only 1-2%. The resulting blood cells were analyzed by an automatic ACT Dif blood counter (Beckman Coulter, Krefeld, Germany) to establish the conditions for the ELUTRA system.

The leukopheresis products were treated by ELUTRA (Gambro BCT, Lakewood, USA) according to the manufacturer's instructions by a counterflow centrifugal elutriation method using a fixed rotor speed (2400 rpm) and the stepwise adjustment of the media flow followed by stopped rotor collection. Then 5000 mL of development buffer was prepared containing Hanks buffered saline (Biochrom, Berlin, Germany) with 1% human serum albumin (Octalbin®, Octapharma, Langen,

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Germany). The ELUTRA process resulted in five fractions, with monocytes enriched in the fraction of the stopped rotor. The cellular composition of the fractions was characterized by an ACT Diff automatic blood meter (Beckman Coulter, Krefeld, Germany) and FACS analysis.

FACS analysis of ELUTRA fractions

Cells from the original leukopheresis product and the five ELUTRA fractions were incubated for 30 minutes with the following monoclonal mouse antibodies conjugated to fluorescein isothiocyanate (FITC) and phycoerythrin (PE): IgG isotype controls (clone X-40) , anti-CD14-FITC (clone: MΦP9), anti-CD19-FITC (clone: 4G7), anti-HLA-DR-FITC (clone: L243) (BD Biosciences, Heidelberg, Germany) and anti-CD3-PE ( clone UCHT1), anti-CD56-PE (C5.9), anti-CD16-PE (clone: DJ130c) and as additional control CD14-PE (TÜK4) (Dako Diagnostics, Hamburg, Germany) and anti-CD67-FITC ( clone: 80H3) (Immunotech, Marseille, France). The cells were washed and resuspended in PBS + 2% fetal calf serum (Biochrom, Berlin, Germany). A flow cytometry analysis was performed on a FACS Calibur device using the Cellquest Pro software (BD Biosciences, Heidelberg, Germany).

Generation of immature dendritic cells derived from monocytes from elutriated monocytes

The cells of the stopped rotor fraction or the so-called fraction 5 were then used directly for DC generation if CD14 + cells represented more than 60% of all cells detected by FACS analysis. Fraction 5 cells were collected from the ELUTRA collection bag and washed once with PBS + 0.5% human serum and seeded at 35 x 106/175 cm2 cell culture flask (NUNC, Wiesbaden, Germany) in 35 mL of a DC medium containing RPMI 1640 with very low amount of endotoxins (Biochrom, Berlin, Germany), 1.5% human serum (mixture of AB-positive adult men) (Blood Bank of the University of Tübingen, Germany) and 10 μg / mL of gentamicin (Biochrom, Berlin, Germany) and were grown for six days at 37 ° C and 5% CO2 in a humidified atmosphere. On day 1, 3 and 6 cell cultures were supplemented with 100 ng / mL of GM-CSF (Leukine® by Berlex, Richmond, USA) and 20 ng / mL of recombinant human IL-4 (R&D Systems, Wiesbaden, Germany) in 7 mL of freshly prepared DC medium per flask.

Dendritic cell maturation

Maturation processes were induced by adding different combinations of cytokines and other reagents, as indicated, to immature DCs on day 6, along with 7 mL of additional freshly prepared DC medium defined herein by flask:

Jonuleit Cocktail: 10 ng / mL of TNF-α, 10 ng / mL of IL-1-β, 15 ng / mL of IL-6 (R&D Systems, Wiesbaden, Germany) and 1 µg / mL of prostaglandin E2 (Minprostin ®, Pharmacia / Pfizer, Erlangen, Germany),

Kalinski cocktail: 10 ng / mL of TNF-α, 10 ng / mL of IL-1-β (R&D Systems, Wiesbaden, Germany), 3000 IU / mL of IFNα (Roferon A®, Roche, Welwyn Garden City, England ), 1000 IU / mL of IFNγ (Imukin®, Boehringer Ingelheim, Ingelheim, Germany) and 20 ng / mL of double stranded RNA (poly I: C, InVivogen, Toulouse, France).

New cocktail 1: 10 ng / mL of TNF-α, 10 ng / mL of IL-1-β (R&D Systems, Wiesbaden, Germany), 5000 IU / mL of IFNγ (Imukin®, Boehringer Ingelheim, Ingelheim, Germany), 1 µg / mL of R848 (InVivogen, Toulouse, France) and 250 ng / mL of prostaglandin E2.

As a variation of cocktail 1, the same components were used except that the IFNγ concentration was reduced to 1000 IU / mL and that of prostaglandin E2 to 100 ng / mL.

New cocktail 2: similar to cocktail 1 plus 20 ng / mL of double stranded RNA (poly I: C, InVivogen, Toulouse, France).

As a control, a flask received only 7 mL of freshly prepared medium and served as immature DC (data not shown).

Collection of dendritic cells

After incubation of the DC with the ripening cocktails for 24 hours, the cells were collected by washing them twice with PBS + 0.5% human serum with gentle agitation, the cells were counted in a Neubauer chamber and prepared for analysis.

Flow cytometric analysis (FACS)

DC phenotyping:

DCs were labeled with the following fluorescein-conjugated monoclonal mouse antibodies with specificities for isotype controls (clone X-40), CD14 (FITC, MΦP9), CD19 (FITC, clone: 4G7), CD86 (FITC, clone: 2331 FUN-1), CD80 (PE, clone: L307.4) (BD Biosciences, Heidelberg, Germany) and CD209 (PE, clone: DCN46) (Pharmingen, San Diego, USA) and CD3 (FITC, clone: UCHT1), CD56 (FITC, clone: C5.9a), CD1a (FITC, clone: NA1 / 34)

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(Dako, Hamburg, Germany) and HLA-DR (PE, clone: B8.12.2), CD40 (PE, clone: mAb89), CD83 (PE, clone: HB15a) (Immunotech, Marseille, France).

CCR7 staining was performed with a rat hybridoma BLR-2 (clone 8E8) (E. Kremmer, GSF) compared to the isotype control for IgG2a of the EBNA-A2 hybridoma (clone R3) by incubation of DC in liquids culture supernatants for 60 minutes, followed by subsequent washing and detection with secondary mouse antibody against rat IgG conjugated with cyanine 5 (Jackson Immuno, West Grove, USA).

To analyze the vitality, the DCs were sedimented and resuspended for 20 minutes in 7-aminoactinomycin D (Sigma-Aldrich, Deisenhofen, Germany) at final concentrations of 10 μg / mL in PBS + 2% fetal calf serum. After washing, the cells were analyzed in the third channel of the FACS Calibur machine.

Maturity stability check (Washing separation test)

The matured, collected and washed DCs were seeded again at 2.5-3 x 106/9 mL of freshly prepared DC medium without cytokines in 25 cm2 cell culture flasks (NUNC, Wiesbaden, Germany).

After approximately 44 hours, the DCs were collected and phenotyped by FACS analysis.

Signal Test 3

DC were co-cultured with cells that mimicked T lymphocytes as described previously (Kalinski, 2004). In summary, the mature DC collected and washed, were seeded again on 96-well plates at concentrations of 2 x 104 / well and incubated together with mouse fibroblasts stably transfected with human CD40L (Garrone P, Neidhardt EM, Garcia E, Galibert L, van Kooten C, Banchereau J. Fas ligation induces apoptosis of CD40-activated human B lymphocytes. J. Exp. Med. 1995 Nov 1; 182 (5): 1265-1273) at concentrations of 5 x 104 / well. To control the proliferation of each cell population alone, DCs without additions and CD40L fibroblasts were analyzed in standard medium. After 24 hours, the plates were centrifuged and the duplicated 8-well supernatant liquids were pooled for IL-10 and IL-12p70 analysis by ELISA.

ELISA (IL-12p70 / IL-10)

The secretion of IL-12p70 and IL-10 was detected by the DCs during the maturation process (primary DCs) and the DCs from the signal 3 assay by standard quantitative ELISA. ELISA was performed using sets of antibody pairs previously analyzed for the detection of IL-12p70 and IL-10 (R&D Systems, Wiesbaden, Germany) according to the manufacturer's instructions. The reaction of the colorimetric substrate with tetramethylbenzidine and H2O2 was measured after stopping with H3PO4 at 450 nm and wavelength correction at 620 nm and analyzed by easy adjustment of the computer program (SLT, Crailsheim, Germany).

Cryopreservation of dendritic cells

After collection and washing, 20-25 x 106 DC was collected in 0.5 mL of cold 20% human serum albumin (Octalbine®, Octapharma, Langen, Germany) and mixed gently with 0.5 mL (equal amounts ) of freshly prepared freezing solution containing 10% glucose (Braun, Melsungen, Germany), 20% DMSO (CryoSURE®, WAK-Chemie, Dessau-Thornau, Germany) in 20% human serum albumin. The cryotubes (NUNC, Wiesbaden, Germany) were stored overnight at -80 ° C and transferred to the gas phase of a liquid nitrogen vessel.

Mixed lymphocyte reaction

DCs were matured in vitro as indicated, washed twice in PBS + 0.5% human serum, irradiated with 40 Gy and grown in 96-well round bottom microplates at 1 x 104 / well (NUNC , Wiesbaden, Germany) in RPMI 1640 + 1.5% human serum. The cryopreserved cells of fraction 3 after the ELUTRA procedure of different donors were used as a source of responding cells and seeded at 1 x 105 / well in the DC of allogeneic donors.

As a control for the activation of T lymphocytes by means of the differences in the main histocompatibility complex (MHC), one third of the cells were used as follows: as stimulant cells a mixture of mononuclear cells (MNC) from 5 independent donors obtained was used of the leukocyte layer after irradiation of 40 Gy. The general nonspecific proliferation potential of T lymphocytes was monitored by incubation of IL-2 responsive cells (Proleukin® from Chiron, Emeryville, USA) at 50 IU / mL and phytohemagglutinin at 10 μg / mL (Sigma-Aldrich, Deisenhofen, Germany).

After 6 days, the cells were pulsed with 0.5 μCi / well of 3 H-thymidine (Amersham-Pharmacia, Freiburg, Germany) and the absorption of 3 H-thymidine was determined after 24 hours using a β counter device (Wallac , Freiburg, Germany).

EGFP-RNA transfection in DC

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EGFP-RNA was produced in vitro and subjected to electroporation in mature DC at 24 hours as described above (Nair, SK, Boczkowski, D., Morse, M., Cumming, RI, Lyerly, HK and Gilboa, E. (1998). Induction of primary carcinoembryonic antigen (CEA) -specific cytotoxic T lymphocytes in vitro using human dendritic cells transfected with RNA. Nat. Biotechnol. 16: 364-369. Javorovic, M., Pohla, H., Frankenberger , B., Wolfel, T. and Schendel, DJ (2005). RNA transfer by electroporation into mature dendritic cells leading to reactivation of effectormemory cytotoxic T lymphocytes: a quantitative analysis. Mol. Ther. 12: 734-743), with the except that each 0.4 cm electroporation cuvette contained a total volume of 300 µL, including 8 µg of EGFP-RNA and 3 x 106 of the DC. After electroporation, the DCs were returned to their original ripening media and incubated in a 24-well plate at 37 ° C and 5% CO2 for 24 or 48 hours before analysis by flow cytometry.

ELISPOT assay of virus specific T lymphocyte activation

For activation, ELUTRA fraction 3 lymphocytes were cultured at 1 x 10 6 cells / well with 1 x 10 5 DC virally loaded with peptides in 24-well plates, in RPMI 1640 medium with 10% human serum; on the third day, 30 IU / mL of IL-2 was added and lymphocytes were collected on the seventh day. Peptides that bound HLAA * 0201 included: CMVpp65495-503 (NLVPMVATV), EBV-BMLF1280-288 (GLCTLVAML), influenza protein 58-66 M1 (GILGFVFTL) or the CEF mixture (PANATecs GmbH, Tubinga, Germany) containing two additional peptides, EBV-LMP-2426-434 (CLGGLLTMV) and influenza RNA polymerase PA46-54 (FMYSDFHFI). In vitro activated T lymphocytes and autologous monocytes plus CEF peptides were incubated in RPMI 1640 medium containing 2 mM L-glutamine, 1 mM sodium pyruvate, penicillin / streptomycin (100 U / mL), 10% AB human serum ( BioWhittaker, Verviers, Belgium) and 20 IU / mL of IL-2 at 37 ° C with 5% CO2 for 24 hours. An IFNγ-ELISPOT analysis was performed as described (Becker, C., et al., (2001). Adoptive tumor therapy with T lymphocytes enriched through an IFNγ capture assay. Nat. Med. 7: 1159-1162. Pohla , H., et al., (2000). Allogeneic vaccination for renal cell carcinoma: Development and monitoring. Bone Marrow Transplant. 25: 83-87), with the exception that PVDF plates previously coated with antibodies (Mabtech AB, Nacka, Sweden) and alkaline streptavidin phosphatase and a BCIP / NBT solution plus ready-to-use substrate (Mabtech). The points were counted using the AID ELR03 reader system with software 3.2.3 (AID Autoimmun Diagnostika GmbH, Strassberg, Germany).

2. Results and conclusions

Primary DC culture

ELUTRA fraction 5 of the described example (DC034) contained: 80.6% of CD14 + cells and the following contaminants, 2.89% of CD3 cells, 2.2% of CD56, 1.47% of CD19 and 7.72 % of CD67 + and was therefore appropriate for generating dendritic cells.

The greatest recovery of dendritic cells, based on total seeded cells as well as monocytes (CD14 + cells), was found using Jonuleit cocktail, while the lowest was found using Kalinski cocktail (Figure 1A). A low number of cells represents little capacity for DC collection without using cell scrapers or enzymatic digestion. This finding represents a great disadvantage of the Kalinski cocktail in our system that could be due to a higher degree of adhesion and a fine stretching of the dendritic veils of the DC matured with this cocktail. The viability of the collected dendritic cells determined by staining with 7AAD showed more than 97% of living cells using the Jonuleit cocktail while the cells matured with the Kalinski cocktail reached only 79.5%, the new cocktails of the invention ranged between these two values (Figure 1A).

The expression of co-stimulatory molecules, such as CD80 and CD86, reflected the presence of antigen presenting cells, particularly dendritic cells. Figure 1B shows a high level of expression of these molecules in all DC matured with different cocktails. CD14 is a monocytic molecule, but under the influence of GM-CSF / IL-4 and ripening cocktails quickly disappear from the surfaces of dendritic cells. It is shown herein that the DC generated with all cocktails release the expression of CD14 as an evident proof of a differentiation in the direction of the DC, rather than the possible differentiation in the direction of the macrophages.

On the other hand CD83 serves as the most important marker to indicate the state of maturation of the DC. The expression of CD83, in combination with the almost undetectable expression of CD14, showed that in the five matured DC populations, the cells had a DC identity and were highly mature (Figure 1B).

Chemokine receptor 7 (CD197) indicates a migratory potential of DCs to the lymph nodes along with chemokine gradients of CCL19 and CCL21 in the high endothelial venules. All different matured DC populations expressed CCR7 at high levels (Figure 1C).

DC after separation by washing of ripening cytokines (Washing Separation Test)

The stability of the maturation state is an important characteristic of clinically applicable DC, because patients with malignant diseases frequently show high serum cytokine titres

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inhibitors (for example, IL-10, TGF-beta, IL-6). These cytokines can influence injected DCs by reversing them to an immature state and causing a patient's immune system to tolerate vaccinated tumor antigens.

To analyze whether the new cocktails of the invention induced stable maturation, important DC marker molecules were analyzed after all cytokines were washed and the DCs were incubated at least 40 hours after a new seeding in medium alone. Notably, lower viability was found in DC matured with the Kalinski cocktail (Figure 2A). Figure 2A shows, as expected, that viability is lost over time, because DC and cells in that state are depleted after more than 60 hours of incubation with the maturation cocktail have elapsed. suitable for therapy. Only for reasons of the test, the surface expression of certain molecules, such as re-induction of CD14, was checked, which would indicate a reversal of immature DC. As Figure 2B shows, the five different matured DCs expressing very low levels (less than 1%) of CD14 retain while high levels of the CD83 marker for DC maturation. Once again the DC matured with the Kalinski cocktail showed slightly lower CD83 values than the DC matured with the other cocktails.

Figure 2C shows the stability of high levels of expression of the co-stimulatory molecules, CD80 and CD86, and of maintaining the migratory potential by the expression of CCR7.

DC after freezing and thawing

At this stage, the authors of the present invention sought a method for generating a high number of DCs that are then cryopreserved.

It may be conceivable to freeze monocytes or alternatively, freeze whole mature DCs, even after antigen loading. Figure 3 shows the viability and maturation markers of the different DCs matured after the processes of freezing, conservation within the gaseous phase of liquid nitrogen and thawing. A viability of more than 80% is acceptable, which was obtained by DC matured with the Jonuleit cocktail and the new cocktails of the invention (Figure 3A), but not by DC matured with the Kalinski cocktail and cocktail 5 ( containing both poly (I: C)). The CD83 and CCR7 markers of the DCs were expressed at high levels and only very low numbers of CD14 cells were detected in all thawed DC populations (Figure 3B and C).

DC Stimulatory Capacity

To analyze the functional capacities of DC, the authors of the present invention used a mixed lymphocyte reaction with DC as stimulatory cells against allogeneic T lymphocytes. To control the vital capacities of T lymphocytes, the induction of proliferation against a maximum number of different MHC molecules (third part = 5 mixed donors of MNC), mitogenic stimuli (PHA) and the cytokine IL-2 stimulator was analyzed. T lymphocytes Figure 4 shows these controls for autologous T lymphocytes (4A) and an illustrative responder of allogeneic T lymphocytes (4B). Figure 4C shows irradiated DC without responding lymphocytes and it was found that the assay only determined proliferation by responding lymphocytes. Figure 4D indicates the low induction of autologous T lymphocytes compared to an allogeneic responder (Figure 4E).

In Figure 4F, the proliferation of 3 independent allogeneic responding T lymphocytes after co-incubation with different matured DCs was compared compared to autologous T lymphocytes of the DC donor. As expected from the differences in MHC between the 3 respective responding T lymphocytes and DC, a high stimulatory capacity was observed. Again, the DC matured with the Kalinski cocktail showed no stimulation levels comparable to the other matured DC, which may be due to the lower viability and a higher percentage of dead cells during the test procedure.

Release of IL-12p70 and IL-10 from DC

During the differentiation and maturation processes, the DCs secrete cytokines in their culture supernatant liquid. Figure 5A shows the secretion of biologically active IL-12p70 (filled bars) and IL-10 (empty bars) in primary DC cultures. DCs matured with the Jonuleit cocktail do not secrete detectable amounts of IL-12p70, while the Kalinski cocktail, as well as the new cocktails of the invention, induce IL-12p70 at intervals of ng per mL. These results indicate a capacity of the composition to induce cytokine production in DC. In Figure 5B, it is visible that after a simulated encounter of the DC with the potential T lymphocytes within the lymph nodes by means of the CD40 ligand, it is still possible to re-induce IL-12p70 of the matured DCs in the Kalinski cocktail and to a lesser extent also using the new cocktails of the invention.

IL-10, as a potential Th2 cytokine, counter-regulates the polarization of Th1. Taking this effect into account, the inventors calculated the values of both important regulatory cytokines that have a theoretically equal biological potential and determined the IL-12p70 / IL-10 ratio. Figure 5C represents the results that show that Kalinski's cocktail is superior to Jonuleit's, as described in the literature,

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but the new cocktails of the invention also showed positive ratios for the re-induction of IL-12 after 24 hours of binding to CD40 and therefore secreted more IL-12p70 than DC matured with the Jonuleit cocktail.

To summarize the results, a new combination of substances is described herein, including a TLR7 / 8 ligand and other cytokines and supplements, which is capable of inducing complete maturation of CDs and inducing Th1 regulatory abilities in these cells. . Compared with the maturation of DC with the Jonuleit cocktail, the cocktails of the invention also showed high cell viability after harvesting and freezing, high levels of maturation by the expression of CD83, co-stimulatory molecules and migratory potential for CCR7 expression as well as maturation stability. In contrast to the Jonuleit cocktail, the cocktail of the invention is capable of inducing the secretion of IL-12p70 in the primary culture as well as after the simulating interaction with T lymphocytes by binding to CD40. The mature DCs generated with the new cocktail of the invention a combination of the best characteristics of the Jonuleit cocktail in addition to increasing the inducing capabilities of Th1 by means of IL-12p70. In contrast to the DCs obtained after maturation with the Kalinski cocktail, the new cocktails of the invention result in DC with secretion of IL-12p70 without the negative impact of the loss of cell number caused by prolonged cell death processes. .

The methods of the present invention for generating human dendritic cells are compatible with the standards of good manufacturing practice (GMP) and are therefore useful for clinical application in vaccine generation, which could promote Th1 polarization of T lymphocytes. effectors against tumor antigenic structures.

Protein expression after transfer of RNA to DC by electroporation

Various sources of antigens have been considered for use in vaccines for DC-based tumors. RNA is an attractive candidate to provide whole proteins to DCs for treatment and presentation, thus avoiding the need to know specific MHC binding peptides. To analyze the ability of DCs to express the protein after loading it with RNA transcribed in vitro, the expression of the EGFP was analyzed by flow cytometry after transfer of the corresponding RNA. DC3 cells were not included because cocktail 3 was identical to cocktail 4, except for smaller amounts of IFNγ and PGE2 (see, for example, the legend of Fig. 1). It was previously found that EGFP expression was maximal 12-24 hours after RNA transfection to DC1 cells and that expression was stable for 48 hours (Javorovic, M., Pohla, H., Frankenberger, B., Wolfel, T. and Schendel, DJ (2005). RNA transfer by electroporation into mature dendritic cells leading to reactivation of effector-memory cytotoxic T lymphocytes: a quantitative analysis. Mol. Ther. 12: 734-743), therefore, 24 and 48 hours after electroporation, the percentages of EGFP positive cells and average fluorescence intensities (MFI) were measured. DC1 and DC4 cells expressed EGFP while DC2 cells did not express EGFP and DC5 cells did not express (Fig. 6) or only at very low levels EGFP (data not shown). This same pattern was observed at 48 hours (data not shown). The TLR3 ligand, poly (I: C), was present in the Kalinski cocktail and cocktail 5 (DC2 and DC5 cells, respectively), but was absent in the Jonuleit cocktail (DC1 cells) and in cocktail 4 ( DC4 cells). The Kalinski cocktail also contained IFNα, which was not present in any other cocktail. Interestingly, it was found elsewhere that DC matured only with IFNα also did not express the EGFP protein after RNA transfer (Frankenberger, B., et al., (2005). Cell-based vaccines for metastatic renal cell carcinoma: genetically -engineered tumor cells and monocyte-derived dendritic cells. World J. Urol. 3: 166-174).

Induction of the secretion of IFNγ by T lymphocytes with DC pulsed by peptides

Because DC5 cells could not be loaded with RNA, their ability to present peptides was analyzed as an alternative (Fig. 7). Autologous lymphocytes of the ELUTRA fraction 3 of an HLA-A * 0201-positive donor were stimulated for 7 days with DC pulsed with matured peptides in 3-5 cocktails compared to the Jonuleit cocktail. The cells were then re-stimulated for 24 hours with autologous monocytes plus CEF peptides and analyzed in an IFNγ-ELISPOT assay. Specific peptide responses were found with all DC populations, demonstrating that DC5 cells could present peptides for T lymphocyte activation.

Therefore, the authors of the present invention have found that the presence of poly (I: C) in ripening cocktails prevented DCs from expressing the protein after loading it with exogenous RNA, presumably through activation by TLR3 of mechanisms to protect cells from a foreign RNA (Kato, H. et al., (2006) Differential roles of MDA5 and RIG-I helicases in the recognition of RNA viruses. Nature 441: 101-105). Therefore, DC matured in Kalinski's medium or in cocktail 5 cannot be used for RNA-based vaccines, although both are suitable for use with peptides, as shown herein for cocktail 5 and has been previously published for the Kalinski cocktail (Mailliard, RB et al., (2004). Alpha-type-1 polarized dendritic cells: a novel immunization tool with optimized CTL-inducing activity. Cancer Res. 64: 5934-5937). In contrast, cocktails 3 and 4 would be very suitable for generating DC that produce IL-12p70 using peptides or RNA as sources of tumor-associated antigens for the development of a cancer vaccine.

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Interferon-gamma response of autologous lymphocytes stimulated with mature DC.

Lymphocytes from a donor with HLA-A * 0201 were stimulated with autologous matured DC using the Jonuleit cocktail, the Kalinski cocktail and the three new cocktails described in the patent.

The peripheral blood lymphocytes (PBL) were subjected to elutriation and the contents were separated in fraction 3, in which 54.8% of the cells were CD3 + T lymphocytes and 17.7% CD56 + cells, which is characteristic of natural killer cells (NK). PBLs from fraction 3 of elutriation were incubated directly with the different DC populations matured using the different cocktails and after 24 hours their interferon-gamma secretion (IFN-gamma) was measured in a standard ELISPOT assay.

For ELISPOT analysis, ELUTRA fraction 3 PBLs were cultured in triplicate 50 µL per well on pre-coated PVDF antibody plates (Mabtech AB, Nacka, Sweden), and then the plates were incubated for 2 hours at 37 ° C in RPMI 1640 culture medium supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, penicillin / streptomycin (100 U / mL) and 10% human AB serum (BioWhittaker, Verviers, Belgium) to block non-specific binding. The capture antibody was IFNγ-specific clone 1-D1K (Mabtech). DC populations were carefully added to the wells. For baseline evaluation, only DC and lymphocytes were plated. The total culture volume was 150 µL and the plates were incubated in a humidified incubator at 37 ° C with 5% CO2 for 24 hours. After cell separation and thorough washing with PBS / 0.5% Tween 20, incubation was performed with the biotinylated detection antibody, clone 7-B6-1 (Mabtech), and the development of the points as It has been previously described (1,2), with the exception that streptavidin-alkaline phosphatase and a substrate solution plus BCIP / NBT ready for use were used. The points were counted using the AID ELR03 reader system with the software version 3.2.3 (AID Autoimmun Diagnostika GmbH, Strassberg, Germany).

For activation, ELUTRA fraction 3 lymphocytes were cultured at 1 x 106 cells / well with 1 x 105 DC loaded with viral peptides in 24-well plates, in RPMI 1640 medium with 10% human serum; on the third day, 30 IU / mL of IL-2 was added and on the seventh day the lymphocytes were collected. Peptides that bound HLA-A * 0201 included: CNWpp65495-503 (NLVPMVATV), EBV-BMLF1280-288 (GLCTLVAML), influenza protein 58-66 M1 (GILGFVFTL) or the CEF mixture (PANATecs GmbH, Tubinga, Germany) containing two additional peptides, EBV-LMP-2426-434 (CLGGLLTMV) and influenza RNA polymerase PA46-54 (FMYSDFHFI). In vitro activated T lymphocytes and autologous monocytes with or without CEF peptides were incubated in RPMI 1640 medium containing 2 mM L-glutamine, 1 mM sodium pyruvate, penicillin / streptomycin (100 U / mL), 10% human AB serum (BioWhittaker, Verviers, Belgium) and 20 IU / mL of IL-2 at 37 ° C with 5% CO2 for 24 hours. An IFNγ-ELISPOT analysis was performed as described (Becker, C., et. Al. (2001). Adoptive tumor therapy with T lymphocytes enriched through an IFNγ capture assay. Nat. Med. 7: 1159-1162; Pohla , H., et al. (2000). Allogeneic vaccination for renal cell carcinoma: Development and monitoring. Bone Marrow Transplant. 25: 83-87), with the exception that PVDF plates previously coated with antibodies were used for the detection ( Mabtech AB, Nacka, Sweden) and alkaline streptavidin phosphatase and a substrate solution plus BCIP / NBT ready for use (Mabtech). The points were counted using an AID ELR03 reader system with software 3.2.3 (AID Autoimmun Diagnostika GmbH, Strassberg, Germany).

The dominant interferon-gamma producing cells detected at this early time (see Figure 8) are activated NK cells, in contrast to previous experiments, in which the responses represent those of peptide-specific T lymphocytes stimulated by DC during the activation period of 7 days (see Figure 7). This analysis showed that the activation of NK cells was potentiated approximately three times using DC matured in the DC5 cocktail, compared to the DC1, DC3 and DC4 cocktails.

Combined, these studies show that DC, and in particular those of cocktail 5, are capable of activating natural killer cells (Figure 8), as well as effectively re-stimulating peptide-specific effector T lymphocytes that recognize epitopes derived from cytomegalovirus, virus of Epstein-Barr and influenza virus.

3. Summary of the example

Dendritic cell-based (DC) vaccines frequently use DCs derived from mature monocytes with a cytokine cocktail (Jonuleit) of IL-1β, TNFα, IL-6 and prostaglandin E2 (PG). To obtain the DCs that direct T lymphocytes to Th1 responses, the authors of the present invention have sought cocktails that provide DC that produce biologically active IL-12p70. After elutriation of the apheresis products by ELUTRA, monocytes enriched with GM-CSF and IL-4 were cultured for 6 days in a medium with human serum that complied with the GMP. Immature DCs were matured for 24 hours with several cocktails, containing TLR7 / 8 ligands with or without poly (I: C) and interferon-γ, PG, IL-1β and TNFα. Matured DCs expressed> 80% of CD83, CD86, CD80 and HLA-DR, CD40,> 60% of CD209 (data not shown), <2% of CD14 and> 60% of CCR7 chemokine receptor that is housed in the lymph nodes The DCs retained all maturity and expressed typical surface markers after cryopreservation and after separation by washing the cytokines and new culture for 44 hours.

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IL-12p70 and IL-10 were present in DC supernatants matured with cocktails containing TLR7 / 8 ligands. A cocktail of IFNγ, IL-1β, TNFα, PG and the R848 ligand of TLR7 / 8 provided DC secreting IL-12p70 after harvesting and 24 hours of co-culture with CD40L transfected fibroblasts, simulating lymphocyte encounter T in the lymph nodes. The relative amounts of

5 IL-12p70 versus IL-10 and it was found that the DC of the present invention only revealed a slightly lower ratio between IL-12 and IL-10 as documented by Kalinski (IL-1β, TNFα, IFNα, IFNß, poly (I: C)).

The functionality of the matured DCs with the new cocktails of the invention was analyzed by mixed lymphocyte culture and ELISPOT analysis. The DC of the invention induced alterations and stimulated the specific T lymphocytes of viral antigens comparable to the DC generated by the Jonuleit cocktail (data not shown).

10 In summary, this new cocktail for the maturation of DC combines the characteristics of a good harvest, reasonable recoveries, stability of the ripening markers and inducing capacity of Th1 with the procedures of good manufacturing practices (GMP) required for vaccines of High quality DC

Claims (13)

1. A method for in vitro maturation of at least one immature dendritic cell, which comprises stimulating said immature dendritic cell with TNFα, IL-1β, IFNγ, an agonist of TLR7 / 8 and prostaglandin E2 (PG). 2. The method of claim 1, further comprising stimulating said immature dendritic cell with a TLR3 agonist, preferably poly (I: C).

3.

The method of any one of claims 1 or 2, wherein said substances are part of a composition added to the culture medium of said immature dendritic cell.

Four.

The method of any one of claims 1 to 3, wherein said TLR7 / 8 agonist is an immune response modifying compound of the imidazoquinoline type, preferably wherein said compound

The immune response modifier of the imidazoquinoline type is 4-amino-2-ethoxymethyl-α, α-dimethyl-1Himidazol [4,5-c] quinoline-1-ethanol (R848). 5. The method of any one of claims 1 to 4, wherein said immature dendritic cell is an immature dendritic cell derived from monocytes, preferably derived from human peripheral blood mononuclear cells, monocytes, other myeloid progenitor cells, or CD34 + progenitor cells. , 15 by differentiation in vitro to CD14 + cells, or wherein said immature dendritic cell is obtained directly from peripheral blood. 6. The method of any one of claims 1 to 5, wherein the immature dendritic cell is obtained by incubating human peripheral blood mononuclear cells, monocytes or other myeloid progenitor cells with GM-CSF and IL-4 or IL-13. The method of any one of claims 1 to 6, wherein the immature dendritic cell is of human origin. 8. The method of any one of claims 1 to 7, comprising the following steps: a) preparing mononuclear cells from peripheral blood, preferably wherein said mononuclear cells are obtained by peripheral blood leukopheresis, B) incubating the mononuclear cells of step a) with GM-CSF and IL-4 or IL-13, preferably wherein said incubation lasts from 1 to 9, preferably from 2 to 9, more preferably from 2 to 6 days, c) incubating the cells obtained in step b) with a cocktail comprising TNFα, IL-1β, IFNγ, a TLR7 / 8 agonist, prostaglandin E2 (PG) and, optionally, a TLR3 agonist, preferably poly (I: C), wherein said incubation lasts 12 hours to 72 hours, preferably 20 hours or 24 hours, and 30 d) collect the mature dendritic cell or cells. 9. The method of any one of claims 1 to 8, wherein the mature dendritic cell or cells are further loaded in vitro with one or more antigens, preferably wherein said antigen or antigens are supposed to cause maturation of effector T lymphocytes within of the lymph nodes, more preferably where said loading is performed by incubating the mature dendritic cell or cells with the Less a peptide of said antigen or transfecting the dendritic cell or cells with RNA or DNA encoding the antigen. 10. A population of mature dendritic cells, obtainable by the method of any one of claims 1 to 6, 8 or 9, wherein the immature dendritic cell is of human origin and wherein TNFα is used at a concentration of 1 ng / mL at 50 ng / mL, IL-1β is used at a concentration of 1 ng / mL at 50 ng / mL, IFNγ is used 40 at a concentration of 500 U / mL at 10,000 U / mL, the TLR7 / 8 agonist is used at a concentration of 0.2 µg / mL at 5 µg / mL, PGE2 is used at a concentration of 50 ng / mL at 5000 ng / mL and the TLR3 agonist is used at a concentration of 10 ng / mL at 50 ng / mL. 11. A pharmaceutical composition comprising the mature dendritic cell population of claim 10. The population of mature dendritic cells of claim 10, for use in the treatment of a disease selected from the group consisting of oncogenic diseases and infectious diseases. 13. A combined in vitro use of TNFα, IL-1β, IFNγ, a TLR7 / 8 agonist, prostaglandin E2 (PG) and, optionally, a TLR3 agonist, preferably poly (I: C), for the preparation of al less a mature dendritic cell. 18

14.

The use of claim 13, wherein said TLR7 / 8 agonist is an imidazoquinoline-type immune response modifying compound, preferably 4-amino-2-ethoxymethyl-α, α-dimethyl-1Himidazole [4,5-c] quinoline-1-ethanol (R848).

fifteen.

A composition comprising TNFα, IL-1β, IFNγ, a TLR7 / 8 agonist, prostaglandin E2 (PG) and, optionally, a TLR3 agonist, preferably poly (I: C), wherein preferably said TLR7 / 8 agonist it is a compound that modifies the immune response of the imidazoquinoline type, preferably 4 amino-2- ethoxymethyl-α,α-dimethyl-1H-imidazol [4,5-c] quinoline-1-ethanol (R848).

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